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In this study we used information from the three-dimension structure of astacin, the x-ray crystal structure of astacin in complex with a transition state inhibitor 20and the primary structures of different members of the astacin family to identify residues in BMP-1 that might account for the procollagen C-proteinase activity of BMP We wanted to know whether BMP-1myc cleaved procollagen at the physiological site.
Totowa, NJ This approach is made possible by the fact that the metalloproteinase domain of BMP-1 shares high sequence homology with astacin, whose x-ray crystal structure is known Related Content Load related web page information.
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Undigested procollagen P ; containing disulfide-bonded chain migrates near the top of 26273 SDS-gel. Residue numbers are labeled with an asteriskin blocks of 10 residues and specific for BMP-1 and astacin. You’ll be in good company. In preliminary studies we performed a multiple sequence alignment of 31 members of the astacin family of metalloproteinases using MultAlin 25 data not shown.
Briefly, a DNA fragment was amplified using the Xcm Forward 22637 and the antisense mutant primer, and an overlapping fragment was amplified using the sense mutant primer and the downstream Blp Reverse primer. In all the known substrates of BMP-1 the scissile bond resides between a small side-chained residue and an aspartic acid. Figure 3 Cleavage of type I procollagen by recombinant BMP-1myc analyzed under non-reducing conditions. The observation that the culture medium of cells transfected with the E94A mutant did not contain detectable PCP activity also validated the use of COS-7 cells as a good model cell system in which to express recombinant BMP Bold indicates homologous substitutions.
Dashed lineshydrogen bonds; green linesputative disulfide bonds. Mature denotes the mature form of BMP In astacin the active site zinc is pentacoordinated by three 226237, a unique tyrosine residue in the Met turnand a water molecule. However, the fact that the C65A mutant was well secreted raised the possibility that another cysteine residue could substitute for Cys 65 in bonding to Cys In the absence of structural information of the metalloproteinase domain of BMP-1 we reasoned that a valid approach was to examine the function of individual residues by site-directed mutagenesis.
Furthermore, the culture medium from COS-7 cells transfected with the empty vector contained no immunoreactive proteins, which shows that es endogenous levels of BMP-1 were very low. Cys 63 and Cys 66had been mutated were also well secreted. The costs of publication of this article were defrayed in part by the payment of page charges.
Sequence Analysis In preliminary studies we performed a multiple sequence alignment of 31 members of the astacin family of metalloproteinases using MultAlin 25 data not shown.
Furthermore, Western blots using the preimmune rabbit serum were blank data not shown. Glu 94 In astacin the active site zinc is pentacoordinated by three histidines, a unique tyrosine residue in the 22637 turnand a water molecule.
A minor change was that laser densitometry of film exposed to dried gels was replaced by image plate quantitation of the 14 C-labeled proteins. A typical result is shown in Fig. View this article with LENS. This loop contains Lys Lane 114 C-labeled type I procollagen 0. This raised the possibility that Cys 85 could form a disulfide bond with a cysteine other than Cys Medium harvested from COS-7 cells transfected with the empty vector contained no immunoreactive proteins.
The neoepitope anti-peptide antibody detected only the mature enzyme, which occurred in the culture medium and not in the cell lysate.
The importance of Cys 85 in stabilizing the structure da the metalloproteinase domain of BMP-1 was confirmed when C85A was found to be poorly secreted. Latent denotes the latent form of BMP In brief, a peptide corresponding to the 10 N-terminal residues of the mature BMP-1 protein after removal of the prodomain was conjugated to keyhole limpet hemocyanin and subsequently used to immunize two separate rabbits.
The culture media of the cells were examined by Western blot analysis using 22637 9E10 monoclonal antibody. In this study we have used site-directed mutagenesis to identify residues in the metalloproteinase domain of BMP-1 that are important for its PCP activity.
The pcDNA3 vector containing the cDNA for Fs was transfected into COS-7 cells, and the conditioned culture medium and the da lysate were analyzed by Western blotting using the 9E10 antibody which detects the c-myc tag at the C terminus of the molecule and the neoepitope antibody which was raised to a peptide corresponding to the 10 residues at the N terminus of mature BMP Plasmids were extracted with a Qiaprep spin miniprep kit Qiagen.
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The Dz and C65A mutants migrated exclusively as the slower migrating reduced form. The Xcm I site is located at nucleotide Two of these, Cys 63 and Cys 66might form a vs bond with each other The only candidates were Cys 63 and Cys We propose 2627 the disulfide-bonded side chain of Cys 66 is fundamental to the PCP activity of BMP-1, because it backs onto the P1 residue in procollagen. Subsites are shown as arched lines. Assay of Procollagen C-proteinase Recombinant BMP-1 was assayed for procollagen C-proteinase activity using human 14 C-labeled type I procollagen substrate and analysis of the cleavage products on SDS gels as described 3.
Western blot analysis showed that the antibody recognized the C-propeptides after cleavage of procollagen with recombinant BMP-1 and BMP-1myc data not shown. The procollagen content was determined by the sensitive hydroxyproline assay method of Terlink